The cell cycle consists of a cell division phase and the events that occur during the period between successive cell divisions, known as interphase. Most cell components are made continuously throughout interphase. As such; it is difficult to define distinct stages in the progression of the growing cell through interphase. One exception is DNA synthesis, since the DNA in the cell nucleus is replicated only during a limited portion of interphase. This period is denoted as the S phase (S=synthesis) of the cell cycle. The other distinct stage of the cell cycle is the cell division phase, which includes both nuclear division (mitosis) and the cytoplasmic division (cytokinesis) that follows. The entire cell division phase is denoted as the M phase (M=mitotic). The period between the M phase and the start of DNA synthesis, i.e., the S phase, is called the G1 phase (G=gap). The period between the completion of DNA synthesis and the next M phase is called the G2 phase. Nuclear membrane dissolution generally takes place immediately prior to cell division, during the M phase or the G2/M interphase. Thus, interphase is composed of successive G1, S, and G2 phases. The duration of an entire cycle varies from cell type to cell type, but is often about 24 hours.
The cell contains exquisitely sensitive feedback control circuits that regulate entry into, exit out of and the events that occur during a given phase of the cell cycle. These circuits can, for example, prevent exit from the S phase if a fraction of a percent of DNA remains unreplicated, and they can block advance into anaphase in mitosis until all the chromosomes have aligned on the metaphase plate. The progression of a cell through the mitotic cycle is controlled by an array of regulatory factors that act as “checkpoints” and assure that the previous stage has been completed before the subsequent stage ensues. The relative abundance of these factors oscillates as the cell cycle advances, either by synthesis of a particular gene product or by chemical transformation, such as phosphorylation and dephosphorylation events. In particular, the progression of the cell to the next stage of its cycle is positively regulated by a family of master enzymes, i.e., cyclin-dependent kinases (see, Sherr, Cell, 73:1059-1065 (1993)). These enzymes are composed of two proteins, a regulatory subunit (the cyclin) and an associated catalytic subunit (the actual cyclin-dependent kinase or CDK), the levels of which vary with different phases of the cell cycle (see, Peters, G., Nature, 371:204-205 (1994)). Both cyclins and CDKs represent molecular families that encompass a variety of genetically related, but functionally distinct proteins.
Numerous protocols and reagents are used to synchronize cells in specific stages of the cell cycle. For example, cycling cells can be synchronized at a specific stage of the cell cycle by growth factor deprivation(see, Keyomarsi, et al., Cancer Res, 51:3602-3609 (1991). PCT Publication No. WO 94/00095 discloses the use of various calpain inhibitors to synchronize the cell cycle. Synchronization of cells, by itself, is a research tool that is not known to be useful for treating disease in a patient.
It is known, however, that compounds that induce cell cycle arrest (e.g., hydroxyurea, VM-26, cisplatin and taxol) can be used therapeutically for cancer treatment. The general theory behind this use is that cancer cells are among the most rapidly proliferating cells in multicellular organisms, and that they would therefore be more susceptible to compounds that disrupt or arrest cell cycling. Moreover, it is a developing medical practice to employ combinations of conventional drugs, including cell cycle blocking agents, for the treatment of cancer. Various combinations of conventional drugs known in the art are as follows: for acute lymphocytic leukemia—vincristine, prednisone, doxorubicin and L-asparaginase; for Hodgkin's disease—mechoroethamine, vincristine, procarbazine and prednisone (MOPP); for histiocytic lymphoma—cyclophosphamide, vincristine, procarbazine and prednisone (C-MOPP); and for testicular carcinoma—bleomycin, vinblastine, and cisplatin.
Although combinations of conventional drugs are being used to treat diseases, no combinations employ a combination of conventional drugs together with the newly developed gene drugs, i.e., expressible nucleic acids, therapeutic genes, etc.
Gene drugs have become available in recent years for the treatment of various types of diseases. A fundamental hurdle for gene drugs is delivery of the gene to the target or treatment site because large nucleic acids are rapidly degraded upon exposure to serum. However, this hurdle has been overcome for local and regional (i.e., inhalation or direct injection) delivery by the use of viral vectors, lipid complexes and the like. For systemic (i.e., intravenous) delivery, the hurdle has been overcome by the use of stable plasmid-lipid particles (“SPLPs”) disclosed in PCT Publication No. WO 9640964, which is assigned to the assignee of the instant invention and which is incorporated herein by reference. SPLPs are fully encapsulated lipid-plasmid particles that are resistant to nuclease degradation, have low immunogenicity, and are of small size (<150 nm), thereby making them particularly well suited for long circulation lifetimes. Therapeutic uses for these SPLPs have been disclosed in U.S. patent applications Ser. Nos. 60/063,473; 60/073,598; 60/082,665; 60/086,917, all of which are assigned to the assignee of the instant invention and are incorporated herein by reference.
Son and Huang, Proc. Natl. Acad. Sci., 91:12669-12672 (1994), reported that improved expression of a plasmid delivered directly to tumor cells can be obtained in tumor cells seven days after treatment with cisplatin. Other drugs, including vincristine, were not found to be effective. Son and Huang did not teach, suggest or appreciate the benefits associated with the synchronization of cells at the treatment site for enhanced use of therapeutic gene drugs. Further, Son and Huang neither taught not appreciated that the synchronization of cells could be used to increase the efficiency of transformation, nor did they teach or suggest that synchronized cells at certain stages of the cell cycle are more efficiently transformed with therapeutic genes.